The human multidrug resistance protein family is composed of a number of well characterized members (See, e.g., Borst et al., 2000, J Natl Cancer Inst 92:1295-1302). Originally implicated in the resistance of tumor cells to chemotherapeutic agents, the multidrug resistance protein P-glycoprotein (P-gp), an ATP-driven transmembrane efflux pump and a product of the multiple drug resistance 1 (MDR1) gene, belongs to the ATP-binding cassette (ABC) family of proteins. P-gp is an ATP-dependent drug transporter that is predominantly found in the apical membranes of a number of epithelial cell types in the body, including the luminal membrane of the brain capillary endothelial cells that make up the blood-brain barrier. While humans have only one drug-transporting P-gp (MDR1), mice have two, mdr1a (also called mdr3) and mdr1b (also called mdr1) (Gros et al., 1986, Cell 47:371-380; Hsu et al., 1989, J Biol Chem 264:12053-12062; Devault et al., 1990, Mol Cell Biol 10:1652-1663). The tissue distributions of these proteins suggest that the mouse isoforms together perform the same function(s) as the single human MDR1 protein. P-gp is expressed in the human intestine, blood-brain barrier, liver, kidneys, testes and other tissues (Matheny et al., 2001, Pharmacology 21(7):778-796; Nolnar et al., 1997, Anticancer Res 17(1A):481-486; Bradbury, 1993, Exp Physiol 78:453-472; Sugawara, 1990, Acta Pathol Jpn 40:545-553; Cordon-Cardo et al., 1990, J Histochem Cytochem 9:1277-1287).
Expression of P-gp, localized to cell membranes may affect the bioavailability and biodistribution of drug molecules that are substrates for this transporter. Drugs that inhibit P-gp can alter the absorption, metabolism, disposition, and elimination of co-administered drugs and can enhance bioavailability or cause unwanted drug-drug interactions.
Over the last decade, a large number of structurally diverse compounds has been shown to be transported out of cells by P-gp, leading to a much lower availability of these compounds in their intended tissues than would be expected from the physical properties of the compounds (Schinkel et al., 1996, J Clin Invest 97:2517-2524) which only share the properties of being small (usually <2 kDa) hydrophobic amphipathic molecules that are usually not negatively charged.
Classes of small molecule therapeutics that are substrates of P-gp include anticancer, immunosuppressive, cardiac, anti-histamine and a number of anti-infective types including compounds effective against human immunodeficiency virus (HIV). The activity of P-gp also decreases the intracellular availability of a variety of anticancer drugs, leading to the development of resistance to them. The same appears to be true for HIV protease and non-nucleoside reverse transcriptase inhibitors (Fellay et. al., 2002, Lancet 359:30-36). For example, P-gp limits the intestinal absorption of digoxin, talinolol and cyclosporine after oral dosing, limits the central nervous system penetration of human immunodeficiency virus protease inhibitors, and excretes paclitaxel (taxol) into the intestine (Lown et al., 1997, Clin Pharmacol Ther 62:248-260; Sparreboom et al., 1997, Proc Natl Acad Sci USA 94:2031-2035; Kim et al., 1998, J Clin Invest 101:289-294; Schwarz et al., 2000, Int J Clin Pharmacol Ther 38:61-167). Apical expression of P-gp in tissues such as liver, kidney and intestine results in reduced drug absorption from the gastrointestinal tract and enhanced drug elimination into bile and urine. Moreover, expression of this glycoprotein in the endothelial cells of the blood-brain barrier prevents entry of certain drugs into the central nervous system.
The major documented cause of the multidrug resistance of cancers is the overexpression of P-gp, which is capable of pumping structurally diverse anti-tumor drugs from cells (Houseman et al., A Molecular Genetic Approach to the Problem of Drug Resistance in Chemotherapy, 504-517 (1987) (Academic Press, Inc. ); Fine and Chabner, Multidrug Resistance, in Cancer Chemotherapy 8,117-128 (Pinedo and Chabner eds. 1986)). Increased expression of the gene encoding P-gp is found in many malignant cells, including leukemia, lymphoma, sarcoma and carcinoma (Cordon-Cardo et al., 1990, J Histochem Cytochem 9:1277-1287). Active P-gp is believed to function as a “hydrophobic vacuum cleaner” which expels hydrophobic drugs from targeted cells. Such drugs include many anti-cancer drugs and cytotoxic agents, such as vinca alkaloids, anthracyclines, epipodophyllotoxins, taxanes, actinomycins, colchicine, puromycin, toxic peptides (e.g., valinomycin), topotecan, and ethidium bromide (See, Pastan and Gottesman, 1987, New England J Med 316(22):1388-1393). Thus, tumor cells expressing elevated levels of the multiple drug transporter accumulate far less anti-tumor agents intracellularly than tumor cells having low levels of this transporter. The degree of resistance of certain tumor cells has been documented to correlate with both elevated expression of the drug transporter and reduced accumulation of the anti-tumor drugs (Gottesman and Pastan, 1988, J Biol Chem 263,12163; Fojo et al., 1985, Cancer Res 45:3002).
Noninvasive, nuclear imaging techniques can be used to obtain basic and diagnostic information about the physiology and biochemistry of living subjects in general, including experimental animals, normal humans and patients, and for P-gp function in particular, including experimental animals, normal humans and patients. These techniques, including PET (positron emission tomography) and SPECT (single photon emission computed tomography) rely on the use of imaging instruments that can detect radiation emitted from radiotracers administered to living subjects. The information obtained can be reconstructed to provide planar and tomographic images that reveal the distribution and/or concentration of the radiotracer as a function of time.
PET is a noninvasive imaging technique that offers the highest spatial and temporal resolution of all nuclear medicine imaging modalities and has the added advantage that it can allow for true quantitation of tracer concentrations in tissues. The technique involves the use of radiotracers, labeled with positron-emitting radionuclides, that are designed to have in vivo properties that permit measurement of parameters regarding the physiology or biochemistry of a variety of processes in living tissue. SPECT is a nuclear medicine tomographic imaging technique using gamma rays arising from administered radiotracers. It is very similar to conventional nuclear medicine planar imaging using a gamma camera. However, SPECT is able to provide true 3D information.
Radiotracers can be labeled with positron- or gamma-emitting radionuclides. The most commonly used positron-emitting radionuclides are 15O, 13N, 11C and 18F, which are usually accelerator-produced and have a half life of 2, 10, 20 and 110 minutes, respectively. The most widely used gamma-emitting radionuclides are 18F, 99mTc, 201TI and 123I.
Several radiotracers have been developed for PET that are ligands for specific neuroreceptor subtypes such as [11C]raclopride and [18F]fallypride for dopamine D2/D3 receptors, [11C]WAY-100635 for serotonin 5-HT1A receptors, [11C]McN 5652 and [11C]DASB for serotonin transporters, [18F]altanserin and [3H]ketanserin for serotonin 5-HT2A receptors, or enzyme substrates (e.g., 6-FDOPA for the enzyme, aromatic amino acid decarboxylase) (e.g., Ehrin et al., 1985, Int J Appl Radiat Isot 36(4):269-73; Mukherjee et al., 1997, Synapse 27(1):1-13; Suehiro et al., 1993, Life Sci 53(11):883-92; Houle et al., 2000, Eur J Nucl Med. 27(11):1719-22; Meyer et al., 2004, Am J Psychiatry 161(5):826-35; Simon et al., 2007, NeuroImage 34:1317-1330; Lemaire et al., 1991, J Nucl Med 32(12):2266-72; Biver et al., 1997, Nucl Med Biol 24(4):357-60; Pike et al., 1995, Eur J Pharmacol 283(1-3):R1-3; Gunther et al., 1995, Nucl Med Biol 22(7):921-7). These agents permit the visualization of neuroreceptor or enzyme pools in the context of a plurality of neuropsychiatric and neurologic illnesses.
A number of PET and SPECT radiotracers have been developed to demonstrate the presence of P-gp in tissue, but none of these are applied to drug development or currently used as routine clinical diagnostic tool (Del Vecchio et al., 2000, Cancer Biother Radiopharm 15:327-337; Hendrikse and Vaalburg, 2002, Methods 27:228-233; Levchenko et al., 2000, J Nucl Med 41:493-501). Although these imaging tools have their utility, their sensitivity and therefore their scope for research purposes is limited. At most, a 2-3 fold increase of uptake in the P-gp expressing tissue (brain/tumor) is observed at an assumed 100% inhibition dose. This means that if small changes (e.g., <20%) in P-gp functionality suffice for co-treatment in, for example, tumor therapy, current imaging tools may not be sensitive enough to establish the change in P-gp functionality with sufficient confidence and may therefore not be suitable for establishing the required dose of a P-gp inhibitor or competitive substrate.
The most widely examined radiotracers for P-gp imaging include [11C]colchicine (Levchenko et al., 2000, J Nucl Med 41:493-501), [11C]verapamil (Elsinga et al., 1996, J Nucl Med 37:1571-1575; Takano et al., 2006, J Nucl Med 47:1427-1433), [11C]daunorubicin (Elsinga et al., 1996, J Nucl Med 37:1571-1575; Takano et al., 2006, J Nucl Med 47:1427-1433), [18F]paclitaxel (Kurdziel et al., 2003, J Nucl Med 44:1330-1339), [94mTc]sestamibi (Bigott et al., 2005, Mol Imaging 4:30-39), and [11C]loperamide (Passchier et al., 2003, Mol Imaging Biol 5:121 (abstract); Wilson et al., 2005, J Labelled Compd Radiopharm 48:S142 (abstract)) for PET, and [99mTc]sestamibi (Piwnica-Worms et al, 1993, Cancer Res 53:977-984) for SPECT (Del Vecchio et al., 1997, Eur J Nucl Med 24:150-159). All of these radiotracers suffer from one or more limitations, such as (i) difficult radiosynthesis (e.g., [94mTc]sestamibi), (ii) troublesome metabolism resulting in significant contamination by radiometabolites (e.g., [11C]verapamil), or (iii) low sensitivity, i.e., a low signal to noise ratio because of modest increase of brain uptake after P-gp inhibition. These limitations have so far compromised their use for sensitive and quantitative assessment of P-gp function in vivo, especially in human subjects.
Loperamide, 4-(p-chlorophenyl)-4-hydroxy-N,N-dimethyl-α,α-diphenyl-1-piperidinebutyramide hydrochloride, a synthetic piperidine derivative, is a drug effective against diarrhea resulting from gastroenteritis or inflammatory bowel disease. In most countries loperamide is available generically and under brand names such as Lopex™, Imodium™, Dimor™ and Pepto Diarrhea Control™. Loperamide is an opioid receptor agonist and acts on the μ-opioid receptors in the myenteric plexus large intestines (Awouters et al., 1993, Digestive Diseases and Sciences 38:977-995). Loperamide does not cross the blood-brain barrier in significant amounts. Any loperamide molecules that do cross the blood-brain barrier are quickly exported from the brain by the P-gp (Sadeque et al., 2000, Clin Pharmacol Therapeutics 68:231-237).
Loperamide (free base) has the following formula (XV):

Pharmacokinetic studies have been performed in rats and human using [3H]loperamide labeled at the metabolically unstable methyl groups of the tertiary amide, and in rats using [3H]loperamide labeled at the position adjacent to the chlorine substitution (Heykants et al., 1974, Arzneim.-Forsch 24:1649; Heykants et al., 1977, Eur J Drug Metab Pharmacokinet 2:81-91). Metabolites of loperamide, such as, desmethyl-loperamide (dLop) and didesmethyl-loperamide have been described (Yoshida et al., 1979, Biomedical Mass Spectrometry Vol 6, No. 6. 253-259; Heykants et al., 1977, European Journal of Drug Metabolism and Pharmacokinetics 2:81-91; Miyazaki et al., 1982, Life Sciences 30:2203-2206). Oxidative N-dealkylation, including demethylation, seemed to be the major metabolic pathway.
As described herein, Applicants have performed PET studies in non-human primates using 11C-labeled loperamide having the formula (XVI):
In these studies it was found that [11C]loperamide was heavily metabolized and resulted in several undesirable radiometabolites which precluded any possibility of quantitative analysis of P-gp function with [11C]loperamide and PET.
Modification of P-gp function is an important underlying mechanism of drug interactions in humans; both inhibition and induction of the protein having been reported as the cause of drug-drug interactions. Compounds which act as P-gp substrates potentially have an increased risk of pharmacokinetic problems in man. There is therefore considerable interest in the pharmaceutical field in determining, at an early stage, whether new drug candidates are potential P-gp substrates as this may significantly reduce their biological efficacy. Due to its importance in pharmacokinetics, P-gp transport screening has now become an integral part of the drug discovery process. However, existing technology for quantifying P-gp transport is generally low through-put, labor-intensive and expensive, characteristics which are far from optimal for meeting the demands of high-throughput screening of the pharmaceutical industry.
The P-gp transport system is complex and poorly understood in man in vivo. Highly sensitive radiotracers which could be used in vivo would be especially beneficial in elucidating P-gp's role in drug and toxin resistance, immunity, apoptosis or cell differentiation. The present invention provides effective new radiotracers and methods for imaging P-gp function. One of the radiotracers of the present invention, [11C]N-desmethyl-loperamide, a metabolite of [11C]loperamide, surprisingly was identified by the inventors to also be a P-gp substrate, and superior to [11C]loperamide for imaging P-gp function.